1. Field of the Invention
The present invention relates generally to a method of and device for performing vertical spectrophotometric determinations, and, more particularly, a method of and device for performing vertical spectrophotometric determinations of a substance based on a fixed beam pathlength. The invention further relates to a method of and device for performing spectrophotometric determinations of substances using vertical beam spectrophotometric based instrumentation such as a microplate reader.
2. Description of the Background
The microplate industry has for some time used beam photometry to perform spectrophotometric determinations on liquid samples. This methodology is based on the transmission of a focused light beam through a liquid or gaseous light-absorbing substance contained in a vessel (e.g., a cuvette or microplate well) and the detection of the unabsorbed transmitted light by a light detector to measure the light absorption (or optical density) of the substance, thereby providing a measurement which can be related to the presence and amount of light absorbing matter in the liquid. U.S. Pat. No. 5,073,029 discloses several such opto-electronic devices and is incorporated herein by reference.
In conventional vertical beam photometry for performing spectrophotometric determinations (hereinafter, vertical spectrophotometry) as shown in FIG. 1, a light beam 2 emitted from a light source 1 is transmitted in the vertical direction along a vertical axis of the vessel 3 containing the solution 4 to be analyzed. The light absorption of the solution 4 is then measured by a detector 5 at the opposite end of the vessel positioned to receive the light beam 2 after it passes through the solution 4. Examples of such vertical spectrophotometric equipment include the SPECTRAMAX PLUS and the VMAX KINETIC READER from MOLECULAR DEVICES CORP., and the POWER WAVE 200 and EL310 ELISA MICROPLATE READER from BIO-TEK INSTRUMENTS, INC.
In horizontal beam photometry for performing spectrophotometric determinations (hereinafter, horizontal spectrophotometry) as shown in FIG. 2, the light beam 7 emitted from a light source 6 is transmitted along a horizontal path which is at a right angle to the vertical axis of the vessel 8 containing the solution 4. The absorption of the light is then measured by a detector 9 at the opposite side of the vessel positioned to receive the light beam 7 after it passes through the solution 4. Examples of such horizontal spectrophotometric equipment include the SPECTRONIC GENESYS 5 by SPECTRONICS INSTRUMENTS, INC., and the SHIMADZU UV1201 by SHIMADZU, INC. spectrophotometers.
In horizontal spectrophotometry, the use of specific fixed light pathlength distances (e.g., 1 cm) have become customary in order to directly compare results from different instruments and solutions. This has led to the generation of tabular information relating light absorption measurements with the concentration of specific known substances, thus, allowing the direct determination of the concentration of certain elements in a sample of the tested substance based on its absorption at a specific wavelength of light. Because the vessel containing the substance has a fixed cross sectional dimension (e.g., inner diameter) which sets the light beam pathlength distance, highly precise absorption measurements can be made using horizontal spectrophotometry.
However, unlike horizontal spectrophotometry, in vertical spectrophotometry, as in the case where substances are tested in microplate wells, the distance that the light travels through the light absorbing substance, or the pathlength, varies and is dependent upon the volume of the substance, as well as a number of other factors such as the surface tension properties of the solution which can create variations in the meniscus formed in the vessel. These factors result in a high degree of variability in the light beam pathlength for any given sample substance, and, as a result, the resulting absorption determination can be imprecise. Furthermore, with the increased use of newer vessels having smaller well volumes as in the case of microplates with 364 or more wells, the physical properties of the liquid-solid interaction become more pronounced increasing the error percentage in any given reading using vertical spectrophotometry.
In order to overcome the imprecision associated with vertical spectrophotometry, prior art solutions include treating the inside surface of the vessel with a surfactant in order to minimize problems associated with surface tension, thereby reducing the variability in the meniscus formed in the sample vessel. For example, U.S. Pat. No. 4,741,619 (incorporated herein by reference) discloses making the walls of a microplate hydrophilic by treating them with surfactant so as to reduce the curvature of the meniscus obtained in the center of the wells that is perpendicular to the vertical light beam. Accordingly, the well to well variability of the meniscus is minimized, thereby improving the reliability of vertical beam spectrophotometric measurements.
While treating the well walls with a surfactant may decrease the variability in the meniscus, the process does not eliminate the meniscus effect nor is it compatible with all light-absorbing substances. Furthermore, attempts to reduce variability in the meniscus do not address variability in the total volume of the is sample. Thus, the determination of the exact light beam pathlength, which is critical for the direct conversion of an absorption measurement to a concentration value, is often difficult as a result of the indeterminate liquid surface level.
Other attempts to overcome the problems associated with conventional vertical spectrophotometry include combining vertical and horizontal spectrophotometry to accomplish the task of determining the pathlength of solutions in microplate wells. In these instances a horizontal spectrophotometric measurement is made of a light absorbing solution contained in a vessel of known dimensions and then compared to the determination made from a vertical spectrophotometric measurement. The SPECTROMAX PLUS is an example of a product that performs this task. However, there exist several problems with this comparison methodology. First, the methodology does not prevent variation as a result of surface tension which causes a meniscus to form on the surface of the liquid-air interface. Furthermore, the methodology introduces additional variability by the use of separate optical paths for vertical and horizontal spectrophotometric measurements.
As the microplate industry has grown and developed, the need for more accurate vertical spectrophotometric determinations has increased. The present invention addresses this need for accurately determining the absorption characteristics of a substance using vertical spectrophotometric measurements by performing these measurements on the basis of a known fixed light beam pathlength of the analyzed substance.